While coordinated activities of the sympathetic and neuroendocrine systems are essential for proper maintenance of bodily homeostasis, sustained sympathohumoral activation is highly detrimental, contributing to several prevalent diseases, including heart failure (HF). Despite this evidence, a comprehensive understanding of the basic mechanisms underlying neurohumoral responses both in physiological and pathological conditions is still missing. The hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei play pivotal roles in the generation of sympathohumoral responses, and accumulating evidence supports elevated neuronal activity in these nuclei in animal models of HF. However, the precise underlying mechanisms remain incompletely understood. The atypical gas neurotransmitters, particularly nitric oxide (NO), are recognized as critical inhibitory signaling molecules in the brain, mostly in areas involved in autonomic/neuroendocrine integration. In fact, a blunted NO function has been shown to contribute to sympathohumoral activation in HF. Here, we propose the gas molecule carbon monoxide (CO) as a novel signaling mechanism within the SON/PVN. We obtained preliminary results showing that in opposition to NO, CO stimulates hypothalamic neuronal function. Thus, we put forward the novel concept that a balance between two opposing gas molecules is critical in determining neurohumoral outflows from the hypothalamus. Using a multidisciplinary approach combining in vitro electrophysiology, cell imaging, tract tracing and immunohistochemistry, we will test the central hypothesis that elevated CO bioavailability contributes to exacerbated SON/PVN neuronal activity in HF, and that these effects are mediated by blunting NO inhibitory function. We will test our central hypothesis in 2 specific aims: 1 - To determine the specific cellular sources of CO within the SON/PVN in sham and HF rats. Our working hypothesis is that the CO-synthetizing enzyme heme-oxygenase (HO) is expressed in specific cell populations within the SON/PVN, and that an elevated expression occurs in HF rats. 2 - To determine the cellular targets and mechanisms of action of CO within the SON/PVN in sham and HF rats. Our working hypothesis is that CO is an excitatory gas molecule targeting both neurosecretory and presympathetic neurons. We expect results from this R21 proposal to provide the proof-of-concept that CO is endogenously produced within the SON and PVN, and that it is a functionally relevant gas molecule influencing neuronal activity in these brain regions. We will begin to understand how changes in CO/NO interactions contribute to altered neuronal activity, and consequently neurohumoral output in an animal model of HF. We believe this knowledge will broaden our understanding of basic cellular mechanisms contributing to the hypothalamic control of homeostasis, as well as how changes in these mechanisms lead to pathological process in prevalent human diseases.